Human leukocyte antigens (HLA) are encoded by HLA gene complex located on the short arm of human chromosome six. The human HLA genes are part of the major histocompatibility complex (MHC), a cluster of genes associated with tissue antigens and immune responses. Successful organ transplantation between individuals depends on the degree of acceptance, i.e., histocompatibility, between donor and recipient pairs. Antigens that cause rejection to the transplanted organ are transplantation antigens or histocompatibility antigens. There are more than twenty antigen systems related to rejection reaction in a human body. Among them, the one that can cause strong and acute rejection reaction is called major histocompatibility antigen. Its gene is a cluster of tightly connected genes, called major histocompatibility complex (MHC). It has now being proved that the immune response gene (IR gene) that controls immune response and regulating function is located in MHC. Thus, MHC not only relates to transplantation rejection but also involves widely in induction and regulation of immune response and regulation. HLA genes are located in a region of about 4000 kb located on human chromosome six, occurring about 1/3,000 of the the entire human genome. There are 224 identified HLA loci. The HLA proteins are classified, based on their structures, expression pattern, tissues distribution, and function, into three classes: HLA-I, HLA-II, and HLA-III. Within each gene locus, there are hundreds of alleles.
The proteins encoded by HLA genes play an important role in graft rejection during tissue transplantation. Successful tissue transplantation depends on achieving a degree of HLA matching between donor and recipient. Thus, HLA typing is necessary for selection of an optimally matched donor. Currently, HLA typing is routinely done in connection with many medical procedures, e.g., organ transplantation, especially bone marrow transplantation. Based on extensive polymorphism in HLA genes of the human population, the role of the proteins encoded by HLA genes in regulating immune response, and codominant expression by both the paternal and maternal genes, HLA typing is also used in predicting susceptibility to diseases, forensic identification, paternity determination, and genetic studies. Accordingly, there is a need for accurate HLA typing methods.
Different methods have been used for HLA typing. Currently, HLA genes are typed using serological methods, mixed lymphocyte culture methods (MLC), and DNA sequence-based typing methods.
Serological methods are based on reactions of sera with the HLA proteins on the surface of lymphocytes. Methods based on the principle of serological typing, such as ID-IEF and monoclonal antibody typing method, have been developed to improve specificity and shorten the testing time. Major drawbacks to serological HLA typing are the complexity of the sera, the lack of widespread availability of standard sera necessary to conduct the tests, and that only the already known HLA types, but not new polymorphisms, are detected.
In mixed lymphocyte culture (MLC) tests, lymphocytes from one individual (the “responder”) are cultured with “stimulating” lymphocytes from another individual. When the stimulating cells are from unrelated persons or family members whose MHC is different from that of the responder, the untreated lymphocytes proliferate; this proliferation is an indicator for non-matching antigens from the individuals. MLC methods are not widely used for the lack of availability of typing cells and complexity of testing procedures.
DNA sequence-based HLA typing methods have been developed to overcome drawbacks with serological or mixed lymphocyte culture methods. One such method involves the use of DNA restriction fragment length polymorphism (RFLP) as a basis for HLA typing. See U.S. Pat. No. 4,582,788. Polymorphism in the length of restriction endonuclease digests generated by polymorphism in the HLA genes of the human population in combination with polymerase chain reaction (PCR) technology are used for HLA typing. However, RFLP method fails to differentiate between certain alleles that are known to exist in the population (e.g., subtypes of HLA-DR4), and thus, cannot be used to distinguish certain combinations of alleles. Moreover, its practical usefulness is limited because the procedures involved take about two weeks to complete and require use of radioactivity.
More recently, researchers have established sequence-specific oligonucleotide (SSO) probe hybridization method to perform HLA-II typing. The method entails amplifying a polymorphic region of a HLA locus using PCR, hybridizing the amplified DNA to a sequence-specific oligonucleotide probe(s), and detecting hybrids formed between the amplified DNA and the sequence-specific oligonucleotide probes. This method can identify one or two nucleotide difference between HLA alleles. The drawbacks of this method is the complexity and difficulty of making multiple equivalent membranes for hybridization or reuse of the same membrane after hybridization which currently is not automated due to the high number of alleles under investigation. Although reverse line strip typing method has been developed to improve the SSO method using an enzymatic method for generating signals for detection, the operation of this method is complicated and difficult to get desired results.
Sequence specific primer amplification (PCR-SSP) method for HLA typing utilises the specific sequence sites in PCR primer for PCR amplification of HLA type and analyzes amplified product by electrophoresis. The time required for the test using this method is only 2 to 3 hours. Mytilineos et al., Hum. Immunol., 59: 512-7 (1998). However, for an unknown sample, the method requires a lot of research for testing each specific primer. In addition, it is difficult to obtain high resolution typing for HLA subtypes.
Other DNA sequence-based HLA typing method includes PCR single strand conformation polymorphism (PCR-SSCP) and PCR fingerprinting. DNA sequence-based HLA typing method has made HLA typing more precise and also help identify more HLA alleles.
DNA chip technology has been widely used for analysing a large number of different DNA sequences or fragments simultaneously on a single DNA chip. The technique allows high-throughput, simultaneous and fast analysis of DNA fragments and requires very minute amount of the target DNA fragment. Because of the complexity of HLA genes, DNA chip can be an ideal tool for use in HLA typing. A few kits and methods have been described. See Kahiwase, Rinsho Byori Suppl. 110: 99-106 (1999); Cao et al., Rev. Immunogenet. 1: 177-208 (1999); and Guo et al., Rev. Immunogenet. 1: 220-30 (1999).